The color of the skin is determined by the amount of melanin produced by melanocytes. It is well known that differences in the intensity of color of the skin are not due to anatomical but functional factors, since the number of melanocytes is in all cases identical, irrespectively of the color of the skin, and is genetically determined (Summa Gallicana Vol. 2, Chapter 27). Melanin is produced in the form of granules, which are released through an active mechanism and reach the epidermis, where they result in the skin color. An alteration in the mechanisms of melanin production and release involves a disturbance of melanic pigmentation of the skin. Accordingly, it is possible to stimulate pigmentation of the skin by modifying the above-mentioned mechanisms (Fistarol S, Itin P., Disorders of Pigmentation, J. Dtsch. Dermatol. Ges. 2010; 8(3):187-201).
To obtain a darker complexion with an effect of tanning without sun exposure, or to reinforce the natural tan achieved upon reduced exposure or make it last longer, products to be applied on the skin, generally known as “self-tanning” products are in use These products are often used as an alternative to traditional sun exposure to avoid the risk of consequences associated with excessive sun exposure, such as sunburn, premature skin aging and increased risk of skin cancer.
The products currently used for tanning without sun exposure are based on the reaction of an active chemical agent present in the product with the amino acids of the skin. Various chemical compounds of this type are known, among which dihydroxyacetone (DHA) is the most widespread one. When applied, DHA interacts with dead cells of the stratum corneum of the epidermis, causing a change in color of the surface layer with an effect similar to tanning. The latter is produced from 2 to 4 hours after application and generally lasts five to seven days after the initial application.
Although recommended for use as a safer alternative than direct solar radiation or UV irradiation, the self-tanning agents such as DHA have a number of disadvantages, including the ability to stain on clothes, the ability to give to the skin a non-uniform color and usually a typical unpleasant smell.
Another field where a treatment capable of effectively darkening the skin is required is that of hypopigmentary cutaneous disorders, among which vitiligo is the best known one.
Vitiligo is a fairly common non-contagious skin disease, characterized by a disorder of skin pigmentation, which is manifested by the appearance of irregular and sharply-edged patches, having very variable shape and size, in which the skin is de-pigmented. The de-pigmentation is caused by a loss of the natural pigment of the skin (melanin), with the formation of a light spot, called macula, which spreads over time affecting a larger area of the body.
The de-pigmented patches may appear anywhere in the body, but the most commonly involved sites are hands, arms, the skin of the anogenital, perioral and periorbital regions. The patches edges are often hyperpigmented, which evidences the contrast with the color of normally pigmented surrounding skin. The onset of vitiligo is independent of sex and skin color of those affected, and its appearance is, therefore, as more evident as darker the complexion of the person affected is. Apart from the change in color, the skin of the affected areas is absolutely normal.
The patches may gradually extend over time, sometimes instead they remain steady, but they rarely regress. The course of the disease may be aggravated by psychic trauma and anxiety.
The percent incidence of vitiligo, which seems to arise more frequently between 20 and 40 years of age, but also arises in pediatric age, turns out to be higher in industrialized countries (where it may also reach 3-4% of the population), while the global incidence would be 1%.
The origin of vitiligo is unknown, although autoimmune factors and/or genetic predisposition are suspected. Various pathogenic theories have been formulated, the most reliable ones being the autoimmune theory, the self-cytotoxic theory and the neural or neurogenic theory. None of them has so far been able to satisfactorily explain the appearance of the typical de-pigmented skin patches. The only certain element is that, by examining under a microscope a fragment of skin taken from an achromic area, a reduction of melanocytes and lack of melanin may be observed, melanin being the pigment to which the skin color is due (Kim Y C et al., Histopathologic features in vitiligo, Am. J. Dermatopathol., 2008).
For hitherto practically unknown causes, therefore, the melanocytes are unable to synthesize melanin and can be numerically reduced or replaced by other cells: in this case the cells that will migrate from the basal layer of the epidermis towards the upper layers will be devoid of melanin. These cells, once emerged on the surface, will give rise to the appearance of an achromic patch.
The fastest and most used aesthetic option to deal with skin dyschromias such as vitiligo is the application of cosmetics able to conceal the de-pigmented areas. In cases of particularly large de-pigmentations, topical de-pigmenting products are used, such as hydroquinone or monobenzone, which serve to circumvent the problem, as they eliminate areas of pigment neighboring to achromic patches in order to create a homogeneity of color on the skin.
Since it is generally recognized that melanocytes undergo degeneration as a result of a local inflammation, which may be of autoimmune origin, the main therapy for vitiligo is based on topical immunomodulating agents (in particular, corticosteroids). The latter are applied on the de-pigmented areas, usually in combination with ultraviolet light (UV) irradiation. Other known treatments include the application of activated vitamin D3 or photosensitizing agents (psoralens), again in combination with ultraviolet light (UV) irradiation.
A therapeutic approach currently accepted and widespread at present for the treatment of vitiligo is represented by psoralens therapy in combination with ultraviolet irradiation limited to the UV-A range, also known as PUVA therapy or PUVA-therapy. This has been shown to be able to effectively improve the condition of skin discoloration in approximately 50% of cases, while steroids are moderately effective only in cases of fast-spreading vitiligo, and the disorder often occurs again after the therapy was discontinued.
The last therapeutic resort, when all other treatments have proved to be ineffective or inapplicable, is represented by surgical treatment through healthy skin grafting.
Among the substances that have been found to be potentially capable of increasing skin pigmentation, capsaicin, curcumin and piperine have been particularly studied. These substances are contained, respectively, in chili, curry and black pepper, and may be effective in controlling the progression of vitiligo. The use of these natural antioxidants for the treatment of the disease has emerged, in particular, from a biochemistry survey conducted in vitro on primary cultures of keratinocytes.
It has been found experimentally that some applications of piperine and of some synthetic derivatives thereof to the skin of laboratory mice, coupled or not with phototherapy with ultraviolet rays, made the skin of rodents darker in six weeks. The effect was more evident and durable for the combined treatment with UV irradiation. Such therapeutic use of the piperine is also described in the international patent application publ. No. WO 00/02544 (in the name of BTG International Ltd.), while the U.S. Pat. No. 7,361,685 (assigned to the Oregon Health and Science University) extends such application to a class of piperine derivatives. Consequently, creams containing piperine or also capsaicin as vanilloid skin receptor TRPV1 agonists and modulators of melanogenesis have been proposed for the treatment of cutaneous achromias and hypochromias such as vitiligo.
One of the mechanisms that have been hypothesized analyzing the therapeutic results obtained in the treatment of vitiligo with PUVA is a reduction in local levels of nerve growth factor (NGF). Around the 90 s it had already been suggested that an increased expression of the NGF receptor could be associated with the destruction of skin melanocytes (Yaar M. et al., J. Clin. Invest., 1994), thus resulting in a cutaneous de-pigmentation.
In line with the negative effects of local neuromediators on the skin pigmentation, a reduced local sensitivity has been documented in the skin patches with vitiligo, as well as the presence of nerve endings in a degenerative state (Breathnach A., et al., J. Invest. Dermatol., 1992). In vitro studies have also shown that another neurotransmitter called neuro-tensin may increase the local inflammation and worsen the skin pigmentation in vitiligo through the induction of TNF-α (Tumor Necrosis Factor-α) by the melanocytes (Kovacs S O. J. Am. Acad. Dermatol., 1998).
As it is known, the nerve growth factor is the first component of a complex family of neurotrophins, and is well-known for its trophic, tropic and differentiating action on cholinergic neurons of the central nervous system and on the peripheral sympathetic system. NGF is produced in many mammalian tissues, including human, and is released into the bloodstream at higher levels during the growth and differentiation of the nervous system. Biological, biochemical and molecular studies carried out on in vitro cell systems showed a high sequence homology between murine and human NGF. Furthermore, in humans, as in other animal species, the NGF is normally present both in the cerebrospinal fluid and in blood stream at concentrations in the range of 10 to 15 pg/ml, which increase in some inflammatory pathologies (autoimmune diseases, allergic diseases, etc.) and decrease in others (diabetes)
The NGF was discovered by Prof. Rita Levi-Montalcini, at the Zoology Institute of the Washington University of St. Louis (Levi-Montalcini R., Harvey Lect., 60:217, 1966), and its discovery represented a remarkable advance in the study of the growth and differentiation mechanisms of the nerve cell, as NGF is able to affect the development and preservation of the biological functions of the neurons and their regeneration. For the discovery of this molecule, and for having characterized its biological function both in the peripheral and the central nervous system, in 1986 Prof. R. Levi-Montalcini was awarded the Nobel Prize for Medicine and Physiology.
A number of in vitro and in vivo studies have demonstrated the pathophysiological importance of NGF in preventing neuronal damage of surgical, chemical, mechanical and ischemic nature, making it the ideal candidate for use in the treatment of several conditions of the peripheral and central nervous systems (Hefti F., J. Neurobiol., 25:1418, 1994; Fricker J., Lancet, 349:480, 1997). In fact, since many years ago clinical trials on patients suffering from Parkinson's disease and Alzheimer's disease have been carried out, by intracerebral administration of murine NGF (see, e.g., Olson L. et al., J. Neural Trans.: Parkinson's Disease and Dementia Section, 4: 79, 1992). Results of these studies confirmed the observations made in animal models and showed the absence of possible side effects following administration of murine NGF. This feature was subsequently confirmed for the human recombinant NGF (Petty B. G. et al., Annals of Neurology, 36:244-246, 1994).
Studies on the characterization of biological, biochemical, molecular, preclinical and clinical effects of NGF have been carried out almost exclusively using NGF isolated from submandibular glands of adult rodents. Therefore, the widest amount of acquired data currently concerns murine NGF. The biochemical properties of the latter have been described, in particular, in a work dating back to 1968 (Levi-Montalcini R. e Angeletti P. U., Physiological Reviews, 48:534, 1968).
The NGF contained in murine salivary glands is a 140 kdalton molecular complex, with a sedimentation coefficient equal to 7 S, consisting of three sub-units, α, β and γ, the second one of which represents the actual active form. The latter, called βNGF, with a sedimentation coefficient of 2.5 S, is usually extracted and purified according to three not very different techniques (Bocchini V., Angeletti P. U., Biochemistry, 64:787-793, 1969; Varon S. et al., Methods in Neurochemistry, 203-229, 1972; Mobley W. C. et al., Molecular Brain Research, 387: 53-62, 1986). In turn, the βNGF thus obtained is a dimer consisting of two identical chains of 118 amino acids, having an overall molecular weight of about 26,000 Daltons. Each single chain is stabilized by three disulfide bridges, while non-covalent bonds ensure the dimeric structure formation. This molecule, being very stable, is soluble in almost any solvent, either aqueous or oily, maintaining unchanged its biochemical characteristics and biological activity. Further details about the structure, physical and biochemical characteristics of the molecule are reported in Greene, L. A. e Shooter, E. M., Ann. Rev. Neurosci. 3:353, 1980.
Recently, the βNGF structure has been further clarified by means of crystallographic analysis. This analysis has revealed the presence of three anti-parallel filament pairs, having a β-type secondary structure, capable of forming a flat surface along which the two chains join together to give the active dimer. On these βNGF chains the presence of four “loop” regions has been evidenced, wherein many variable amino acids are located. The specificity of recognition by the receptor is likely to be due to the same.
It is known that the biological effect of NGF is mediated by two receptors present on the surface of the corresponding target cells, namely the high-affinity receptor TrkA (tyrosine kinase A) and the low affinity p75. There are several antibodies which selectively inhibit the biological effect of NGF, the existence of which allowed an accurate characterization and modulation of its action, both in cellular systems and in vivo.
In more recent times it has become possible to synthesize, by using genetic engineering techniques, the human NGF (Iwane, M. et al., Biochem. Biophys. Res. Commun., 171:116, 1990), and small amounts of human NGF have also become commercially available. However, it was found by direct experience that the biological activity of human NGF is very low compared to the activity of murine NGF. Furthermore, it should be kept in mind that almost all of the currently available data in man, both in vitro and in vivo, have been obtained using murine NGF, and that undesirable effects amenable to the murine origin of the molecule have never been experienced.
With reference to a possible involvement of nerve growth factor in pigmentary disorders of the skin, it has been assumed for a long time, as noted above, that the NGF is able to take part in the cited neurogenic inflammation, which in turn is involved in the pathogenesis of various skin conditions (Pincelli C., Eur. J. Dermatol., 2000). Starting from this hypothesis, some scientific reports published in the field of specialistic dermatology refer to a possible use of antagonists of NGF in the treatment of hypopigmentary disorders of the skin (El-Samad Z A. et al., Egypt. J. Derm. & Androl. Vol. 27, No. 3,4, 2006; Lee M H. et al., Korean J. Physiol. Pharmacol., 2002). In particular, it has been found that the PUVA therapy reduces the cutaneous density of nerve endings (Tominaga M. et al., J Dermatol. Sci, 2009), while it is known that the topical application of NGF would produce an opposite effect.
In line with the proposal of the cited authors to use an antagonist of NGF in the treatment of vitiligo, other studies have suggested that a therapy aimed at reducing the skin levels of NGF could be beneficial to the hypopigmentary skin lesions of vitiligo (Rateb A. et al., J. Egypt. Wom Dermatol. Soc, 2004). In this study it is suggested that in hypo-pigmented skin patches an increased expression of NGF occurs, and that the activation of the NGF receptors placed on melanocytes is critical for their degeneration/destruction in vitiligo and that therefore, theoretically, an NGF antagonist may represent a future therapy for vitiligo.
For the treatment of vitiligo, it has also been proposed to use a family of biologically active molecules of natural origin known as Fibroblast Growth Factor (FGF), in particular the basic Fibroblast Growth Factor (bFGF), and polypeptide sequences derived from it. In particular, in the U.S. Pat. No. 6,143,723 and in the patent applications EP-A-1754489 and US 2007/0027080 (all in the name Abburi, Ramaiah) there is proposed the use of preparations containing partial sequences of bFGF for topical application on the skin for the treatment of vitiligo, either alone or in combination with other therapies already known for the treatment of vitiligo.
Recently, the patent application US 2010/0222275, (Tamaki et al.), proposed a topical preparation for the treatment of vitiligo based on an agent having a thickening activity on the epidermis. The patent application listed, as agents having such an activity, agents belonging to the group of Fibroblastic Growth Factor (FGF)—both acid (aFGF) and basic (bFGF)—Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF).